The present invention relates to the method of production of cartilage tissue for surgical implantation into human joints for the purpose of filling defects of the articular cartilage or replacing damaged or degenerated cartilage.
Cartilage Injury and Repair
Human joint surfaces are covered by articular cartilage, a low friction, durable material that distributes mechanical forces and protects the underlying bone. Injuries to articular cartilage are common, especially in the knee. Data from the Center for Disease Control (CDC) and clinical studies have suggested that approximately 100,000 articular cartilage injuries occur per year in the United States. Such injuries occur most commonly in young active people and result in pain, swelling, and loss of joint motion. Damaged articular cartilage does not heal. Typically, degeneration of the surrounding uninjured cartilage occurs over time resulting in chronic pain and disability. Cartilage injuries therefore frequently lead to significant loss of productive work years and have enormous impact on patients"" recreation and lifestyle.
Joint surface injuries may be limited to the cartilage layer or may extend into the subchondral bone. The natural histories of these types of injuries differ. Cartilage injuries which do not penetrate the subchondral bone have limited capacity for healing (1). This is due to properties inherent to the tissue. Nearly 95 percent of articular cartilage is extracellular matrix (ECM) that is produced and maintained by the chondrocytes dispersed throughout it. The ECM provides the mechanical integrity of the tissue. The limited number of chondrocytes in the surrounding tissue are unable to replace ECM lost to trauma. A brief overproduction of matrix components by local chondrocytes has been observed (2); however, the response is inadequate for the repair of clinically relevant defects. Cellular migration from the vascular system does not occur with pure chondral injury and extrinsic repair is clinically insignificant.
Osteochondral injuries, in which the subchondral bone plate is penetrated, can undergo healing due to the influx of reparative cells from the bone marrow (1). Numerous studies have shown, however, that the complex molecular arrangement of the ECM necessary for normal cartilage function is not recapitulated. The repair response is characterized by formation of fibrocartilage, a mixture of hyaline cartilage and fibrous tissue. Fibrocartilage lacks the durability of articular cartilage and eventually undergoes degradation during normal joint use Many osteochondral injuries become clinically asymptomatic for a period of a few to several years before secondary degeneration occurs. However, like isolated chondral injuries, these injuries ultimately result in poor joint function, pain, and disability.
Molecular Organization of the ECM
The physical properties of articular cartilage are tightly tied to the molecular structures of type II collagen and aggrecan. Other molecules such as hyaluronan and type IX collagen play important roles in matrix organization. Type II collagen forms a 3-dimensional network or mesh that provides the tissue with high tensile and shear strength (3). Aggrecan is a large, hydrophilic molecule, which is able to aggregate into complexes of up to 200 to 300xc3x97106 Daltons (4)]. Aggrecan molecules contain glycosaminoglycan chains that contain large numbers of sulfate and carboxylate groups. At physiological pH, the glycosaminoglycan chains are thus highly negatively charged (5). In cartilage, aggrecan complexes are entrapped within the collagen network. A Donnan equilibrium is established in which small cations are retained by electrical forces created by the sulfate and carboxylate groups (6). Water is in turn retained by the osmotic force produced by large numbers of small cations in the tissue.
When the joint is mechanically loaded, movement of water results in perturbation of the electrochemical equilibrium. When the load is removed, the Donnan equilibrium is reestablished and the tissue returns to its pre-loaded state (7). The physical properties of articular cartilage are tightly tied to the molecular structures of type II collagen and aggrecan. Other matrix molecules, such as hyaluronan (8) and type IX collagen (9), play important roles in matrix organization. Failure to restore the normal molecular arrangement of the ECM leads to failure of the repair tissue over time, as demonstrated by the poor long-term performance of fibrocartilage as a repair tissue (10).
Distinct compartments have been demonstrated within the ECM. These differ with respect to the composition and turnover of matrix macromolecules. Immediately surrounding each chondrocyte is a thin shell of ECM characterized by a relatively rapid turnover of matrix components (11). This region is termed the pericellular matrix (11). Surrounding the pericellular matrix is the territorial matrix. Further from the cells is the interterritorial matrix (11). Turnover of matrix macromolecules is slower in the interterritorial matrix than in the pericellular and territorial matrices (11). The role that these various compartments play in the function of the tissue as a whole is unclear. From the perspective of articular cartilage repair, however, they represent a higher level of matrix organization that must be considered in the restoration of injured tissue.
Surgical Treatment of Articular Cartilage Injury
Current methods of surgical restoration of articular cartilage fall into three categories: (1) stimulation of fibrocartilaginous repair; (2) osteochondral grafting; and (3) autologous chondrocyte implantation. Fibrocartilage, despite its relatively poor mechanical properties, can provide temporary symptomatic relief in articular injuries. Several surgical techniques have been developed to promote the formation of fibrocartilage in areas of cartilage damage. These include subchondral drilling, abrasion, and microfracture. The concept of these procedures is that penetration of the subchondral bone allows chondroprogenitor cells from the marrow to migrate into the defect and effect repair. The clinical success rate of this type of treatment is difficult to assess. In published series, success rates as high as 70% are reported at 2 years; however, the results deteriorate with time. At five years post-treatment, the majority of patients are symptomatic.
In osteochondral grafting, articular cartilage is harvested with a layer of subchondral bone and implanted into the articular defect. Fixation of the graft to the host is accomplished through healing of the graft bone to the host bone. The major advantage of this technique is that the transplanted cartilage has the mechanical properties of normal articular cartilage and therefore can withstand cyclical loading. The major disadvantages are donor-site morbidity (in the case of autograft) and risk of disease transmission (in the case of allograft). Additionally, tissue rejection can occur with allografts which compromises the surgical result. Osteochondral autografting (mosaicplasty) has demonstrated short-term clinical success. The long-term effectiveness is unknown. Osteochondral allografts are successful in approximately 65% of cases when assessed at 10 years post-implantation, but are generally reserved for larger areas of damage extending deep into the subchondral bone.
Autologous chondrocyte implantation is a method of cartilage repair that uses isolated chondrocytes. Clinically, this is a two-step treatment in which a cartilage biopsy is first obtained and then, after a period of ex vivo processing, cultured chondrocytes are introduced into the defect (12). During the ex vivo processing, the ECM is removed and the chondrocytes are cultured under conditions that promote cell division. Once a suitable number of cells are produced, they are implanted into the articular defect. Containment is provided by a patch of periosteum which is sutured to the surrounding host cartilage. The cells attach to the defect walls and produce the extracellular matrix in situ. The major advantages of this method are the use of autologous tissue and the ability to expand the cell population. Difficulties with restoration of articular cartilage by this technique fall into three categories: cell adherence, phenotypic transformation, and ECM production.
Cell Adherence
The success of implantation of individual cells (without ECM) is critically dependent upon the cells attaching to the defect bed. Cartilage ECM has been shown to have anti-adhesive properties, which are believed to be conferred by small proteoglycans, dermatan sulfate, and heparan sulfate. Normal chondrocytes possess cell-surface receptors for type II collagen (13) and hyaluronan (11), but it is not clear to what extent ex-vivo manipulated cells possess receptors for these matrix molecules that are functional. An in vitro study of chondrocyte binding to ECMs suggests that the interaction is weak. An in vivo study in rabbits suggests that only 8% of implanted chondrocytes remain in the defect bed after implantation.
Phenotypic Transformation
During the process of expanding the cell population in vitro, chondrocytes usually undergo phenotypic transformation or dedifferentiation (14). Morphologically, the cells resemble fibroblasts. Synthesis of type II collagen and aggrecan is diminished and synthesis of type I collagen, typical of fibrocartilage, is increased. Limited data exist to support the contention that the cells redifferentiate in situ following implantation. Reestablishment of the chondrocytic phenotype is critical to the success of the repair process, as tissue produced by cells which are phenotypically fibroblastic functions poorly as a replacement for articular cartilage.
ECM Production
Prior to implantation, the cultured chondrocytes are enzymatically denuded of ECM. The cells are injected into the defect bed as a suspension. The graft construct is incapable of bearing load and must be protected from weight bearing for several weeks to months. Limited data exist on the quality of the ECM that is ultimately produced. It has been characterized as hyaline-like tissue at second-look arthroscopy two years post-implantation [Petersen, L., personal communication]. The overall recovery period from this form of treatment is 9-12 months. Good or excellent clinical results are achieved in approximately 85% of femoral condyle lesions 2 years post-implantation. However, it is not clear whether the clinical results will be maintained over longer follow-up periods.
Tissue Engineering
Each of the current methods of cartilage repair has substantial limitations. As a result, several laboratory approaches to production of cartilage tissue in vitro have been proposed. These generally involve seeding of cultured cells (either chondrocytes or pluripotential stem cells) into a biological or synthetic scaffold. The major drawbacks of this type of approach are: (1) difficulty in attaining or maintaining the chondrocyte phenotype; (2) unknown biological effects of the scaffold material on the implanted and native chondrocytes and other joint tissues; and (3) limited attachment of the engineered tissue construct to the defect bed.
The present invention involves the production of an implantable cartilage tissue. Its method of preparation and composition address the major problems encountered with current techniques of cartilage repair. The major advantages, features and characteristics of the present invention will become more apparent upon consideration of the following description and the appended claims.
The present invention relates to a transplantable cartilage matrix and a method for its production. Cartilage tissue produced by this method has properties that, with time in culture, become similar to those of a naturally occurring cell-associated ECM. At the time of reimplantation, the matrix in the cartilage tissue has a high rate of turnover (i.e. it is metabolically active). It is rich in cartilage-specific aggrecan proteoglycans and contains enough long hyaluronan chains to allow all these aggrecan molecules to form large aggregates of very large size, but it is relatively poor in collagen pyridinium crosslinks. These properties enhance the implantability of the tissue and subsequent maturation of the tissue in situ following implantation, which leads to integration with the host.
In accordance with the method of the invention, chondrocytes are isolated from tissues containing chondrogenic cells. The isolated chondrogenic cells are cultured in alginate culture for an amount of time effective for allowing formation of a chondrogenic cell-associated matrix. In an important aspect of the invention, the cell-associated matrix has at least about 5 mg/cc3 of aggrecan, a ratio of aggrecan to hyaluronan (mg/mg) between about 10:1 and about 200:1, and a ratio of aggrecan to collagen (mg/mg) between about 1:1 to about 10:1.
Chondrogenic cells, each with a pericellular matrix, are recovered and cultured on a semipermeable membrane system in the presence of serum or serum containing exogenously added growth factor(s). The chondrogenic cells with cell-associated matrix are cultured for a time effective for formation of a cohesive cartilage matrix.
In an important aspect, the invention relates to the use of such in vitro-produced articular tissue in the surgical repair of cartilage damage. Such damage would include acute partial and full thickness chondral injuries, osteochondral injuries, and degenerative processes. Surgical treatment includes open surgical techniques (arthrotomy) and arthroscopic application/insertion of the in vitro-produced cartilaginous tissue.